Amentoflavone induces caspase-dependent/-independent apoptosis and dysregulates cyclin-dependent kinase-mediated cell cycle in colorectal cancer in vitro and in vivo.

Colorectal cancer (CRC) is recognized as the third most common malignancy and the second most deadly in highly developed countries. Although the treatment of CRC has improved in the past decade, the mortality rate of CRC is still increasing. Amentoflavone, one of the flavonoids detected in medical plants, is reported to possess potential anticancer properties in various cancers. However, its role in CRC has not been studied. This study aimed to investigate the role and underlying mechanism of amentoflavone on CRC in vitro and in vivo. We identified the cytotoxicity, apoptosis effect, cell cycle alteration, DNA damage induction and tumor progression inhibition of amentoflavone in HT-29 model by using MTT assay, flow cytometry, immunofluorescence (IF) staining, Western blotting and animal experiments. Amentoflavone induced cytotoxicity is caused by triggering G1 arrest, DNA damage and apoptosis in HT-29 cells. The expression of cyclin D1, CDK4 and CDK6 was decreased by amentoflavone; in contrast, the phosphorylation of ATM and CHK2 and the expression of p21 and p27 were increased. The apoptosis induction of amentoflavone in CRC is not only caspase-dependent but also increases EndoG and AIF nuclear translocation in a caspase-independent manner. Importantly, the apoptosis induction of amentoflavone is not affected by the activity of p53 in CRC. Amentoflavone suppressed the progression of CRC by initiating G1 arrest and ATM/CHK2-mediated DNA damage-responsive, caspase-dependent/independent apoptotic effects. We uncovered a novel tumor-inhibitory role of amentoflavone in CRC that is not associated with p53 activity, which may serve as a potential treatment for CRC.

Evaluation of in vitro cytotoxicity and in vivo potential toxicity of the extract from in vitro cultivated Anoectochilus roxburghii Lindl.

Anoectochilus roxburghii Lind. (A. roxburghii) has promising anti-oxidant, hyperglycemic, hepatoprotective, and immunomodulatory activities as well as anti-tumor effects. However, the pharmacological actions of in vitro cultured plants remain to be determined. Therefore, the objective of the study was to assess in vitro cytotoxicity and in vivo potential toxicity of an extract derived from in vitro cultivated A. roxburghii, termed as iARE. The total flavonoid content and predominant flavonoid compounds of extract were identified and quantitatively analyzed. The in vitro cytotoxicity of iARE was examined using several cancer and normal cell lines. The apoptotic activity and expression of apoptosis-associated genes were also examined in MCF7 cells to determine the underlying mechanisms related to anti-proliferative effects. In vivo potential toxicity of iARE was assessed following acute and subchronic oral administration in Sprague Dawley rats. Quercetin, kaempferol, and isorhamnetin were three flavonoid components identified in iARE. The extract exerted cytotoxic effects on various cancer cells but not normal fibroblasts. Apoptosis in MCF7 cells was induced by iARE in a concentration-dependent manner associated with increased Bax/Bcl-2 ratio and reduced mitochondrial membrane potential ΔΨm, leading to release of cytochrome c, activation of caspase-3/7 and caspase-9, and cleavage of PARP. In the acute oral toxicity study, no mortality or toxicological signs were observed in rats at 1000 or 5000 mg/kg. In a subchronic oral toxicity study, iARE at a dosage of up to 1000 mg/kg produced no mortality or treatment-related adverse effects on general behavior, food intake, body weight, relative organ weights. No apparent marked changes in the histopathology of the liver and kidney were detected. Data demonstrated that iARE induced in vitro cytotoxic effects in cancer cells are associated with lackof invivo toxicity. Thus, iARE was suggested to be considered as apotential therapeutic candidate for cancer treatment.

3-Bromofluoranthene-induced cardiotoxicity of zebrafish and apoptosis in the vascular endothelial cells via intrinsic and extrinsic caspase-dependent pathways.

Fluoranthene, a high-molecular-weight polycyclic aromatic hydrocarbon (PAH), is widely present in air pollutants, including fine inhalable particulate matter. 3-Bromofluoranthene (3-BrFlu), which is a brominated fluoranthene and halogenated PAH, is generated from waste combustion, metallurgical processes, cement production, e-waste dismantling, and photoreaction. Vascular endothelial cells have key functions in the homeostasis and the development of the cardiovascular system. The zebrafish model has been widely employed to study cardiotoxicity and embryotoxicity. However, no evidence has indicated that 3-BrFlu induces cytotoxicity in vascular endothelial cells, or cardiotoxicity and embryotoxicity in zebrafish. In this study, 3-BrFlu induced concentration-dependent changes in embryo- and cardiotoxicity. Cytotoxicity was also induced by 3-BrFlu in a concentration-dependent manner through apoptosis and necrosis in vascular endothelial cells, SVEC4-10 cells. The activities of caspase-3, -8, and -9 were induced by 3-BrFlu via an intrinsic pathway constituting Bcl-2 downregulation, Bad upregulation, and mitochondrial dysfunction; the extrinsic pathway included the expression of death receptors, including tumour necrosis factor α and Fas receptors. These results indicated that 3-BrFlu caused cardio- and embryotoxicity in zebrafish through vascular endothelial cells cytotoxicity resulting from caspase-dependent apoptosis through intrinsic and extrinsic pathways.

Modulated Electro-Hyperthermia Resolves Radioresistance of Panc1 Pancreas Adenocarcinoma and Promotes DNA Damage and Apoptosis In Vitro.

The poor outcome of pancreas ductal adenocarcinomas (PDAC) is frequently linked to therapy resistance. Modulated electro-hyperthermia (mEHT) generated by 13.56 MHz capacitive radiofrequency can induce direct tumor damage and promote chemo- and radiotherapy. Here, we tested the effect of mEHT either alone or in combination with radiotherapy using an in vivo model of Panc1, a KRAS and TP53 mutant, radioresistant PDAC cell line. A single mEHT shot of 60 min induced ~50% loss of viable cells and morphological signs of apoptosis including chromatin condensation, nuclear shrinkage and apoptotic bodies. Most mEHT treatment related effects exceeded those of radiotherapy, and these were further amplified after combining the two modalities. Treatment related apoptosis was confirmed by a significantly elevated number of annexin V single-positive and cleaved/activated caspase-3 positive tumor cells, as well as sub-G1-phase tumor cell fractions. mEHT and mEHT+radioterapy caused the moderate accumulation of γH2AX positive nuclear foci, indicating DNA double-strand breaks and upregulation of the cyclin dependent kinase inhibitor p21waf1 besides the downregulation of Akt signaling. A clonogenic assay revealed that both mono- and combined treatments affected the tumor progenitor/stem cell populations too. In conclusion, mEHT treatment can contribute to tumor growth inhibition and apoptosis induction and resolve radioresistance of Panc1 PDAC cells.

Induction of apoptosis by metformin and progesterone in estrogen-induced endometrial hyperplasia in rats: involvement of the bcl-2 family proteins.

This study compared the antiproliferative effects of metformin and progesterone, via examination of the Bcl-2/Bax-caspase apoptotic pathway in estrogen-induced endometrial hyperplasia (EH) in 40 rats. Two rats died after bilateral oophorectomy, and 1 week after surgery, the remaining 38 were randomly divided into three groups: the first (control, n = 12) received 4 mg/kg 17ß estradiol hemihydrate (E); the second (n = 13) received 4 mg/kg 17ß estradiol hemihydrate and 50 mg/kg metformin (E + M); and the third (n = 13) received 4 mg/kg 17ß estradiol hemihydrate and 1 mg/day medroxiprogesterone acetate (E + MPA). Histological markers and Bcl-2, Bax and caspase 9 expression were analyzed. Luminal epithelial thickness, density of gland and epithelial height was significantly higher in group E than in groups E + M and E + MPA. Histopathologic parameters were similar between the E + M and E + MPA groups. Bcl-2/Bax ratio was significantly decreased in the E + M and E + MPA groups and caspase 9 expression levels were significantly increased in the E + M and E + MPA groups, compared with the control group. In addition, Bcl-2/Bax ratio and caspase 9 expression were similar between the E + M and E + MPA groups. The data indicate that metformin reduces estrogen-induced EH in rats, via activation of the caspase-dependent mitochondrial apoptotic pathway, to the same degree as progesterone.

Cytotoxic and apoptosis-inducing activities, and anti-tumor-promoting effects of cyanogenated and oxygenated triterpenes.

Two of each semisynthetic lanostane- and cycloartane-type triterpenes with a cyano-enone functionality, i.e., 13 and 18, and 23 and 28, respectively, sixteen of their synthetic intermediates, 9-12, 14-17, 19-22, and 24-27, along with seven semisynthetic oxygenated triterpene acetates, 29-35, and eight natural hydroxy triterpenes, 1-8, were evaluated for their cytotoxic activities against leukemia (HL60), lung (A549), stomach (AZ521), and breast (SK-BR-3) cancer cell lines. One natural triterpene, 8, and ten semisynthetic triterpenes, 9, 13, 15, 18, 23, 25, 28, 29, 32, and 33, exhibited potent cytotoxicities against one or more cell lines with IC50 values in the range of 1.4-9.9 µM. Two lanostane-type triterpenes with a cyano-enone functionality, 3-oxolanosta-1,8,24-triene-2-carbonitrile (13) and 3-oxolanosta-1,8-diene-2-carbonitrile (18), induced apoptosis in HL60 cells, as observed by membrane phospholipid exposure in flow cytometry. Western blot analysis showed that 13 and 18 significantly reduced procaspases-3, -8, and -9, and increased cleaved caspases-3, -8, and -9. These findings indicated that compounds 13 and 18 induced apoptosis in HL60 cells via both the mitochondrial and the death receptor-mediated pathways. In addition, upon evaluation of the inhibitory effects on EpsteinBarr virus early antigen (EBV-EA) activation induced with 12-O-tetradecanoylphorbol-13-acetate (TPA) in Raji cells, seven natural triterpenes, 1-6 and 8, and ten semisynthetic triterpenes, 9, 10, 14, 15, 19, 20, 24, 25, 29, and 30, exhibited inhibitory effects which were higher than that of ß-carotene, a vitamin A precursor studied widely in cancer-chemoprevention animal models.

Cytotoxic and melanogenesis-inhibitory activities of limonoids from the leaves of Azadirachta indica (Neem).

Seventeen limonoids (tetranortriterpenoids), 1-17, including three new compounds, i.e., 17-defurano-17-(2,5-dihydro-2-oxofuran-3-yl)-28-deoxonimbolide (14), 17-defurano-17-(2ξ-2,5-dihydro-2-hydroxy-5-oxofuran-3-yl)-28-deoxonimbolide (15), and 17-defurano-17-(5ξ-2,5-dihydro-5-hydroxy-2-oxofuran-3-yl)-2',3'-dehydrosalannol (17), were isolated from an EtOH extract of the leaf of neem (Azadirachta indica). The structures of the new compounds were elucidated on the basis of extensive spectroscopic analyses and comparison with literature. Upon evaluation of the cytotoxic activities of these compounds against leukemia (HL60), lung (A549), stomach (AZ521), and breast (SK-BR-3) cancer cell lines, seven compounds, i.e., 1-3, 12, 13, 15, and 16, exhibited potent cytotoxicities with IC50 values in the range of 0.1-9.9 µM against one or more cell lines. Among these compounds, cytotoxicity of nimonol (1; IC50 2.8 µM) against HL60 cells was demonstrated to be mainly due to the induction of apoptosis by flow cytometry. Western blot analysis suggested that compound 1 induced apoptosis via both the mitochondrial and death receptor-mediated pathways in HL60 cells. In addition, when compounds 1-17 were evaluated for their inhibitory activities against melanogenesis in B16 melanoma cells, induced with α-melanocyte-stimulating hormone (α-MSH), seven compounds, 1, 2, 4-6, 15, and 16, exhibited inhibitory activities with 31-94% reduction of melanin content at 10 µM concentration with no or low toxicity to the cells (82-112% of cell viability at 10 µM). All 17 compounds were further evaluated for their inhibitory effects against the EpsteinBarr virus early antigen (EBV-EA) activation induced by 12-O-tetradecanoylphorbol-13-acetate (TPA) in Raji cells.

Anti-tumor effect and mechanism of the total biflavonoid extract from S doederleinii on human cervical cancer cells in vitro and in vivo.

In this study, the therapeutic effect and possible mechanism of the total biflavonoid extract of Selaginella doederleinii Hieron (SDTBE) against cervical cancer were originally investigated in vitro and in vivo. First, the inhibition of SDTBE on proliferation of cervical cancer HeLa cells was evaluated, followed by morphological observation with AO/EB staining, Annexin V/PI assay, and autophagic flux monitoring to evaluate the possible effect of SDTBE on cell apoptosis and autophagy. Cell cycle, as well as mitochondrial membrane potential (ΔÑ°m), was detected with flow cytometry. Further, the apoptosis related protein expression and the autophagy related gene LC3 mRNA transcription level were analyzed by Western blot (WB) and real-time quantitative polymerase chain reaction (RT-qPCR), respectively. Finally, the anti-cervical cancer effect of the SDTBE was also validated in vivo in HeLa cells grafts mice. As results, SDTBE inhibited HeLa cells proliferation with the IC50 values of 49.05 ± 6.76 and 44.14 ± 4.75 µg/mL for 48 and 72 h treatment, respectively. The extract caused mitochondrial ΔÑ° loss, induced cell apoptosis by upregulating Bax, downregulating Bcl-2, activating Caspase-9 and Caspase-3, promoting cell autophagy and blocking the cell cycle in G0/G1 phase. Furthermore, 100, 200, and 300 mg/kg SDTBE suppressed the growth of HeLa cells xenografts in mice with the mean inhibition rates, 25.3 %, 57.5 % and 62.9 %, respectively, and the change of apoptosis related proteins and microvascular density was confirmed in xenografts by immunohistochemistry analysis. The results show that SDTBE possesses anti-cervical cancer effect, and the mechanism involves in activating Caspase-dependent mitochondrial apoptosis pathway.

Cynanchum paniculatum (Bunge) Kitag. ex H.Hara inhibits pancreatic cancer progression by inducing caspase-dependent apoptosis and suppressing TGF-ß-mediated epithelial-mesenchymal transition.

Background: Cynanchum paniculatum (Bunge) Kitag. ex H.Hara, a member of the Asclepiadaceae family, has a rich history as a traditional Chinese medicinal plant used to treat digestive disorders. However, its potential anti-cancer effects in pancreatic cancer remain largely unexplored. Aim: This study delves into the intricate anti-pancreatic cancer mechanisms of C. paniculatum (Bunge) Kitag. ex H.Hara aqueous extract (CPAE) by elucidating its role in apoptosis induction and the inhibition of invasion and migration. Methods: A comprehensive set of methodologies was employed to assess CPAE's impact, including cell viability analyses using MTT and colony formation assays, flow cytometry for cell cycle distribution and apoptosis assessment, scratch-wound and Matrigel invasion assays for migration and invasion capabilities, and immunoblotting to measure the expression levels of key proteins involved in apoptosis and metastasis. Additionally, a murine xenograft model was established to investigate CPAE's in vivo anti-cancer potential. Results: CPAE exhibited time- and dose-dependent suppression of proliferation and colony formation in pancreatic cancer cells. Notably, CPAE induced apoptosis and G2/M phase arrest, effectively activating the caspase-dependent PARP pathway. At non-cytotoxic doses, CPAE significantly curtailed the metastatic abilities of pancreatic cells, effectively suppressing epithelial-mesenchymal transition (EMT) and downregulating the TGF-ß1/Smad2/3 pathway. In vivo experiments underscored CPAE's ability to inhibit tumor proliferation. Conclusion: This study illuminates the multifaceted anti-proliferative, pro-apoptotic, anti-invasive, and anti-migratory effects of CPAE, both in vitro and in vivo. CPAE emerges as a promising herbal medicine for pancreatic cancer treatment, with its potential mediated through apoptosis induction via the caspase-dependent PARP pathway and MET suppression via the TGF-ß1/Smad2/3 signaling pathway at non-cytotoxic doses. These findings advocate for further exploration of CPAE's therapeutic potential in pancreatic cancer.

Inhibitory effect of a neddylation blockade on HTLV-1-infected T cells via modulation of NF-κB, AP-1, and Akt signaling.

Adult T-cell leukemia (ATL), caused by HTLV-1, is the most lethal hematological malignancy. NEDD8-activating enzyme (NAE) is a component of the NEDD8 conjunction pathway that regulates cullin-RING ubiquitin ligase (CRL) activity. HTLV-1-infected T cells expressed higher levels of NAE catalytic subunit UBA3 than normal peripheral blood mononuclear cells. NAE1 knockdown inhibited proliferation of HTLV-1-infected T cells. The NAE1 inhibitor MLN4924 suppressed neddylation of cullin and inhibited the CRL-mediated turnover of tumor suppressor proteins. MLN4924 inhibited proliferation of HTLV-1-infected T cells by inducing DNA damage, leading to S phase arrest and caspase-dependent apoptosis. S phase arrest was associated with CDK2 and cyclin A downregulation. MLN4924-induced apoptosis was mediated by the upregulation of pro-apoptotic and downregulation of anti-apoptotic proteins. Furthermore, MLN4924 inhibited NF-κB, AP-1, and Akt signaling pathways and activated JNK. Therefore, neddylation inhibition is an attractive strategy for ATL therapy. Our findings support the use of MLN4924 in ATL clinical trials.

Elucidating the development, characterization, and antitumor potential of a novel humanized antibody against Trop2.

Trophoblast cell surface antigen 2 (Trop2) has emerged as a potential target for effective cancer therapy. In this study, we report a novel anti-Trop2 antibody IMB1636, developed using hybridoma technology. It exhibited high affinity and specificity (KD = 0.483 nM) in binding both antigens and cancer cells, as well as human tumor tissues. hIMB1636 could induce endocytosis, and enabled targeted delivery to the tumor site with an in vivo retention time of 264 h. The humanized antibody hIMB1636, acquired using CDR grafting, exhibited the potential to directly inhibit cancer cell proliferation and migration, and to induce ADCC effects. Moreover, hIMB1636 significantly inhibited the growth of MDA-MB-468 xenograft tumors in vivo. Mechanistically, hIMB1636 induced cell cycle arrest and apoptosis by regulating cyclin-related proteins and the caspase cascade. In comparison to commercialized sacituzumab, hIMB1636 recognized a conformational epitope instead of a linear one, bound to antigen and cancer cells with similar binding affinity, induced significantly more potent ADCC effects against cancer cells, and displayed superior antitumor activities both in vitro and in vivo. The data presented in this study highlights the potential of hIMB1636 as a carrier for the formulation of antibody-based conjugates, or as a promising candidate for anticancer therapy.

Natural products and mitochondrial allies in colorectal cancer therapy.

Colorectal cancer (CRC) is a globally prevalent malignancy with a high potential for metastasis. Existing cancer treatments have limitations, including drug resistance and adverse effects. Researchers are striving to develop effective therapies to address these challenges. Impressively, contemporary research has discovered that many natural products derived from foods, plants, insects, and marine invertebrates can suppress the progression, metastasis, and invasion of CRC. In this review, we conducted a comprehensive search of the CNKI, PubMed, Embase, and Web of Science databases from inception to April 2023 to evaluate the efficacy of natural products targeting mitochondria to fight against CRC. Mitochondria are intracellular energy factories involved in cell differentiation, signal transduction, cell cycle regulation, apoptosis, and tumorigenesis. The identified natural products have been classified and summarized based on their mechanisms of action. These findings indicate that natural products can induce apoptosis in colorectal cancer cells by inhibiting the mitochondrial respiratory chain, ROS elevation, disruption of mitochondrial membrane potential, the release of pro-apoptotic factors, modulation of the Bcl-2 protein family to facilitate cytochrome c release, induction of apoptotic vesicle activity by activating the caspase protein family, and selective targeting of mitochondrial division. Furthermore, diverse apoptotic signaling pathways targeting mitochondria, such as the MAPK, p53, STAT3, JNK and AKT pathway, have been triggered by natural products. Natural products such as diosgenin, allopurinol, and clausenidin have demonstrated low toxicity, high efficacy, and multi-targeted properties. Mitochondria-targeting natural products have great potential for overcoming the challenges of CRC therapy.

Inhibition of Caspase 3 and Caspase 9 Mediated Apoptosis: A Multimodal Therapeutic Target in Traumatic Brain Injury.

Traumatic brain injury (TBI) is one of the significant causes of death and morbidity, and it is hence a focus of translational research. Apoptosis plays an essential part in the pathophysiology of TBI, and its inhibition may help overcome TBI's negative consequences and improve functional recovery. Although physiological neuronal death is necessary for appropriate embryologic development and adult cell turnover, it can also drive neurodegeneration. Caspases are principal mediators of cell death due to apoptosis and are critical for the required cleavage of intracellular proteins of cells committed to die. Caspase-3 is the major executioner Caspase of apoptosis and is regulated by a range of cellular components during physiological and pathological conditions. Activation of Caspase-3 causes proteolyzation of DNA repair proteins, cytoskeletal proteins, and the inhibitor of Caspase-activated DNase (ICAD) during programmed cell death, resulting in morphological alterations and DNA damage that define apoptosis. Caspase-9 is an additional crucial part of the intrinsic pathway, activated in response to several stimuli. Caspases can be altered post-translationally or by modulatory elements interacting with the zymogenic or active form of a Caspase, preventing their activation. The necessity of Caspase-9 and -3 in diverse apoptotic situations suggests that mammalian cells have at least four distinct apoptotic pathways. Continued investigation of these processes is anticipated to disclose new Caspase regulatory mechanisms with consequences far beyond apoptotic cell death control. The present review discusses various Caspase-dependent apoptotic pathways and the treatment strategies to inhibit the Caspases potentially.

Temozolomide, Simvastatin and Acetylshikonin Combination Induces Mitochondrial-Dependent Apoptosis in GBM Cells, Which Is Regulated by Autophagy.

Glioblastoma multiforme (GBM) is one of the deadliest cancers. Temozolomide (TMZ) is the most common chemotherapy used for GBM patients. Recently, combination chemotherapy strategies have had more effective antitumor effects and focus on slowing down the development of chemotherapy resistance. A combination of TMZ and cholesterol-lowering medications (statins) is currently under investigation in in vivo and clinical trials. In our current investigation, we have used a triple-combination therapy of TMZ, Simvastatin (Simva), and acetylshikonin, and investigated its apoptotic mechanism in GBM cell lines (U87 and U251). We used viability, apoptosis, reactive oxygen species, mitochondrial membrane potential (MMP), caspase-3/-7, acridine orange (AO) and immunoblotting autophagy assays. Our results showed that a TMZ/Simva/ASH combination therapy induced significantly more apoptosis compared to TMZ, Simva, ASH, and TMZ/Simva treatments in GBM cells. Apoptosis via TMZ/Simva/ASH treatment induced mitochondrial damage (increase of ROS, decrease of MMP) and caspase-3/7 activation in both GBM cell lines. Compared to all single treatments and the TMZ/Simva treatment, TMZ/Simva/ASH significantly increased positive acidic vacuole organelles. We further confirmed that the increase of AVOs during the TMZ/Simva/ASH treatment was due to the partial inhibition of autophagy flux (accumulation of LC3ß-II and a decrease in p62 degradation) in GBM cells. Our investigation also showed that TMZ/Simva/ASH-induced cell death was depended on autophagy flux, as further inhibition of autophagy flux increased TMZ/Simva/ASH-induced cell death in GBM cells. Finally, our results showed that TMZ/Simva/ASH treatment potentially depends on an increase of Bax expression in GBM cells. Our current investigation might open new avenues for a more effective treatment of GBM, but further investigations are required for a better identification of the mechanisms.

Therapy-Induced Tumor Cell Death: Friend or Foe of Immunotherapy?

Combinatory treatments using surgery, radiotherapy and/or chemotherapy together with immunotherapy have shown encouraging results for specific subsets of tumors, but a significant proportion of tumors remains unsusceptible. Some of these inconsistencies are thought to be the consequence of an immunosuppressive tumor microenvironment (TME) caused by therapy-induced tumor cell death (TCD). An increased understanding of the molecular mechanisms governing TCD has provided valuable insights in specific signaling cascades activated by treatment and the subsequent effects on the TME. Depending on the treatment variables of conventional chemo-, radio- and immunotherapy and the genetic composition of the tumor cells, particular cell death pathways are activated. Consequently, TCD can either have tolerogenic or immunogenic effects on the local environment and thereby affect the post-treatment anti-tumor response of immune cells. Thus, identification of these events can provide new rationales to increase the efficacy of conventional therapies combined with immunotherapies. In this review, we sought to provide an overview of the molecular mechanisms initiated by conventional therapies and the impact of treatment-induced TCD on the TME. We also provide some perspectives on how we can circumvent tolerogenic effects by adequate treatment selection and manipulation of key signaling cascades.

A Review on Anti-Tumor Mechanisms of Coumarins.

Coumarins are a class of compound with benzopyrone as their basic structure. Due to abundant sources, easy synthesis, and various pharmacological activities, coumarins have attracted extensive attention from researchers. In particular, coumarins have very significant anti-tumor abilities and a variety of anti-tumor mechanisms, including inhibition of carbonic anhydrase, targeting PI3K/Akt/mTOR signaling pathways, inducing cell apoptosis protein activation, inhibition of tumor multidrug resistance, inhibition of microtubule polymerization, regulating the reactive oxygen species, and inhibition of tumor angiogenesis, etc. This review focuses on the mechanisms and the research progress of coumarins against cancers in recent years.

Ethanolic extract of Cordyceps cicadae exerts antitumor effect on human gastric cancer SGC-7901 cells by inducing apoptosis, cell cycle arrest and endoplasmic reticulum stress.

ETHNOPHARMACOLOGICAL RELEVANCE: Cordyceps cicadae (Miq.) Massee is a traditional Chinese medicine that has been used for approximately 1600 years in China. C. cicadae, a member of the Cordyceps genus, exerts a therapeutic effect on many diseases, such as cancer. OBJECTIVE: This study aimed to evaluate the antineoplasmic activity of C. cicadae and to identify its molecular mechanism of cell death. MATERIALS AND METHODS: The toxicity of the ethanolic extract of C. cicadae (EEC) against different cancer cell lines was determined through MTT assay. Human gastric cancer SGC-7901 cells were treated with EEC for 48 h. Cell morphology was examined by using an Olympus phase-contrast microscope. The cell apoptosis was quantified through Annexin V-FITC/PI staining. Cells were stained with PI and then subjected to flow cytometry for the investigation of cell cycle status. Cells were subjected to mitochondrial membrane potential (MMP) assay after incubation with JC-1 probes and to intracellular Ca2+ measurement through flow cytometry after incubation with Fluo-3 AM fluorescent probes. Western blot analysis was conducted to quantify the expression of proteins related to apoptosis, cell cycle and endoplasmic reticulum stress. High-performance liquid chromatography (HPLC) analysis was performed to analyse the biological activity components of EEC. RESULTS: EEC suppressed the proliferation of SGC-7901 cells and induced the development of abnormal morphological features in a dose-dependent manner. Flow cytometry results indicated that EEC treatment caused cell apoptosis and arrested the cell cycle in the S phase. In addition, EEC treatment triggered MMP depolarization and Ca2+ overloading in the cytosol of SGC-7901 cells. Western blot analysis demonstrated that EEC increased Bax, AIF, caspase-8, caspase-6 and caspase-3 activities and decreased Bcl-2 activity. The release of cytochrome c from mitochondria was associated with mitochondrial dysfunction, which was caused by the activation of the cell surface receptor Fas and the cleavage of PARP. EEC-induced S phase arrest was associated with the up-regulation of E2F1, cyclin A2, cyclin E and p53 expression levels and the down-regulation of CDK2 expression. In addition, EEC increased the expression of endoplasmic reticulum stress-related proteins, such as calpain-1, caspase-12 and caspase-9. HPLC assay results suggested that EEC contained adenine, uridine, adenosine and N6-(2-Hydroxyethyl)-adenosine. CONCLUSION: EEC inhibited the proliferation of SGC-7901 cells by inducing caspase-dependent apoptosis, arresting the cell cycle in the S phase and increasing endoplasmic reticulum stress. This study revealed that C. cicadae is a potential natural source of anticancer drugs.

Modulated electro-hyperthermia induced p53 driven apoptosis and cell cycle arrest additively support doxorubicin chemotherapy of colorectal cancer in vitro.

OBJECTIVE: Modulated electro-hyperthermia (mEHT), a noninvasive complementary treatment of human chemo- and radiotherapy, can generate selective ~42°C heat in cancer due to elevated glycolysis (Warburg-effect) and electric conductivity in malignant tissues. Here we tested the molecular background of mEHT and its combination with doxorubicin chemotherapy using an in vitro model. METHODS: C26 mouse colorectal adenocarcinoma cultures were mEHT treated at 42°C for 2 × 60 minutes (with 120 minutes interruption) either alone or in combination with 1 µmol/L doxorubicin (mEHT + Dox). Cell stress response, apoptosis, and cell cycle regulation related markers were detected using qPCR and immunocytochemistry supported with resazurin cell viability assay, cell death analysis using flow-cytometry and clonogenic assay. RESULT: Cell-stress by mEHT alone was indicated by the significant upregulation and release of hsp70 and calreticulin proteins 3 hours posttreatment. Between 3 and 9 hours after treatment significantly reduced anti-apoptotic XIAP, BCL-2, and BCL-XL and elevated pro-apoptotic BAX and PUMA, as well as the cyclin dependent kinase inhibitor p21waf1 mRNA levels were detected. After 24 hours, major elevation and nuclear translocation of phospho-p53(Ser15) protein levels and reduced phospho-Akt(Ser473) levels were accompanied by a significant caspase-3-mediated programmed cell death response. While mEHT dominantly induced apoptosis, Dox administration primarily led to tumor cell necrosis, and both significantly reduced the number of tumor progenitor colonies 10 days post-treatment. Furthermore, mEHT promoted the uptake of Dox by tumor cells and the combined treatment additively reduced tumor cell viability and augmented cell death near to synergy. CONCLUSION: In C26 colorectal adenocarcinoma mEHT-induced irreversible cell stress can activate both caspase-dependent apoptosis and p21waf1 mediated growth arrest pathways, likely to be driven by the upregulated nuclear p53 protein. Elevated phospho-p53(Ser15) might contribute to p53 escape from mdm2 control, which was further supported by reduced phospho-Akt(Ser473) protein levels. In combinations, mEHT could promote the uptake and significantly potentiate the cytotoxic effect of doxorubicin.

The interaction between the light source dose and caspase-dependent and -independent apoptosis in human SK-MEL-3 skin cancer cells following photodynamic therapy with zinc phthalocyanine: A comparative study.

The aim of this study is to determine the behavior of relative expression of Bcl-2, caspase-8, caspase-9, and caspase-3 genes of/in SK-MEL-3 cancer cells and explore molecular mechanisms responsible for the apoptosis response during an in vitro photodynamic therapy (PDT) with Zinc Phthalocyanine (ZnPc) using different doses of the light source. In this study, firstly the cytotoxic effects of ZnPc-PDT on SK-MEL-3 cells were evaluated. By irradiating the laser, ZnPc induced a significant amount of apoptosis on SK-MEL-3 cells in three IC50s including 0.064±0.01, 0.043±0.01, and 0.036±0.01µg/mL at the doses of 8, 16, and 24J/cm2, respectively. Moreover, flow cytometry and QRT-PCR experiments were done. The high percentage of apoptotic cells was seen in the early apoptosis stage. The expression of Bcl-2 and caspase-8 genes at all doses of laser experienced an obvious reduction in comparison to the control group. On the other hand, although the expression of caspase-9 and caspase-3 genes remains almost constant at 8J/cm2, but they faced an increment at 16 and 24J/cm2 doses. These data reveal caspase-dependent apoptosis in high and caspase-independent apoptosis in low doses of laser. Based on the results of present work, it can be suggested that the dose of the light source is a key factor in induction of caspase-dependent and caspase-independent apoptosis pathways following PDT.

Caspase-dependent and caspase-independent induction of apoptosis in breast cancer by fucoidan via the PI3K/AKT/GSK3ß pathway in vivo and in vitro.

Purpose Fucoidan, a complex, sulfated polysaccharide obtained from brown seaweed, exerted anticancer activity through the down-regulation of ß-catenin signaling in mouse breast cancer cells in our previous study. This study examines the anti-cancer effects of fucoidan as well as its underlying molecular mechanisms in the human triple negative breast cancer (TNBC) cell line and in 7,12-dimethylbenz[a]anthracene (DMBA)-induced experimental mammary carcinogenesis in rats. Methods in vitro studies, fluorescent staining, flow cytometry and Western blotting were performed to analyze apoptosis and protein expression in human breast cancer MDA-MB-231 cells. In vivo intervention experiments were conducted with Sprague Dawley (SD) rats with DMBA-induced breast cancer. Tumor volumes and weights were measured. Results in vitro fucoidan treatment inhibited proliferation and induced apoptosis in MDA-MB-231 cells. Western blotting detected that Cyt C and Smac were released into the cell cytoplasm and that caspase-3 and caspase-9 were activated in MDA-MB-231 cells. The levels of AIF and EndoG were significantly increased in the cytoplasm and in the nuclei by fucoidan. These data show that fucoidan induced caspase-dependent and caspase-independent apoptosis. Moreover, fucoidan treatment down-regulated the expression of Bid, Bcl-2 and Bcl-xl and up-regulated the level of Bax. In vivo, fucoidan supplementation decreased the mean tumor weight. DISCUSSION: Results from the in vivo and in vitro experiments both showed that fucoidan decreased the levels of p-PI3K, p-AKT and p-GSK-3ß (Ser9) in breast cancer. The level of ß-catenin was also decreased. These results suggest that fucoidan can inhibit MDA-MB-231 human breast cancer cells and DMBA-induced tumors in rats by down-regulating the PI3 K/AKT/GSK3ß pathway. This study provides experimental evidence that elucidates the mechanism of antitumor effect of fucoidan and clarifies the mechanism of the effect of fucoidan on the regulation of ß-catenin.